DNA Testing

DNA Tests - A guideline for Canadian Limousin Association Members

DNA Testing Requirements

The CLA bylaws and rules require DNA tests for the following:

- Sire verification for all walking bulls- Sire verification for donor cows- Parent verification and protoporphyria genotype on file for all AI sires (public sales of semen and in-herd use semen)- Parent verification for all embryo calves and Fullblood animals- As of January 1, 2015, all donor cows in embryo production, must have a protoporphyria genotype on file at the Association.

DNA Testing Process

Please follow the below process for requesting DNA testing and sending your samples to the lab.

Every effort is made to ensure DNA test results are returned within 21 calendar days from the time the hair arrives at the lab.

1) Please contact the CLA office to request DNA testing. Animals must be recorded in the CLA registry system Digital Beef before DNA requests can be completed. The CLA staff will need to know the tattoo number(s) of the animal(s) you are requesting DNA testing on. Please indicate whether you require sire verification only or parentage verification and any additional testing (protoporphyria, horned/polled, coat colour) you would like done. You can e-mail limousin@limousin.com or call 403.253.7309 to make your DNA requests to the CLA. Please let the CLA staff know if you require hair collector cards at the time submitting your DNA requests. Hair collector cards are $0.50 each and must be ordered in groups of 10.

2) The CLA staff will generate a DNA application for genotyping for you and e-mail (or fax/mail) this to you. You will need to submit this along with the physical samples to the lab.

3) If you have not done so already, you will need to collect the DNA sample from the animal. It is good practice to collect DNA from all potential sires at the beginning of the breeding season. The most common and easiest form of DNA to collect and submit is hair follicles from the tail switch. The root ends contain the DNA, therefore samples are to be pulled not clipped. Clean the tail switch to remove any foreign material. Comb or brush the tail to remove any dead hair. If needed, wash clean and rinse with water. Wait for the tail to be completely dry. The sample must be free of urine or manure. Dirty samples will not be processed as contamination will make the sample unfit for testing. Wrap approximately 5-10 strands of hair around a finger, about 2 inches away from the skin, and give a sharp pull. Inspect the hair to ensure that the follicles are attached. Hair strands without follicles do not contain DNA and cannot be tested. Repeat until you have obtained approximately 40-50 hair roots. Place the hair sample on the hair card. Please see hair card instructions. Place the sample in the envelope and immediately seal to minimize contamination. Ensure that the envelope has been filled out with the appropriate information identifying what animal the sample came from. Hair from only one animal is to be placed in each envelope. If doing more than one animal, wash hands before starting on the next animal or use a clean pair of surgical gloves. This will reduce the risk of cross-contamination of the samples.

4) Place the application for genotyping (sent to you by the CLA) along with the identified hair sample envelope(s) in a larger envelope and send to Neogen Canada by regular Canada Post mail services:

5) Results will be automatically uploaded into the CLA Digital Beef registry system when they are available. If you are an online user you can view DNA results under the "DNA" tab on each individual animal's profile on Digital Beef. If you are not an online user please contact the CLA office to inquire about DNA results.

DNA Test Options & Cost

Basic SNP Parentage Panel (200 SNPs)

$20

Basic SNP Parentage Panel (500 SNPs)

$23

Basic SNP & Microsatellite Parentage: Sometimes referred to a "SNP transition" or "double testing" - this is required when an animal or it's parent has not been upgraded to SNP technology, the animal must be tested on both microsatellite and SNP panels. The Association staff will inform you if this is ever the case.

Sire or Parentage Verification

Sire verification is required for all walking bulls and for all donor cows. Parentage verification is required for all AI sires (public sales of semen and in-herd use semen), for all embryo calves and for all Fullblood animals. Parentage verification may be completed on a basic SNP panel (200 SNPs) or (500 SNPs), GGP uLD (26,000 SNP) or GGP 50K (50,000 SNP). Testing must be requested through the CLA.

Protoporphyria (Proto) Testing

Protoporphyria is a rare autosomal recessive genetic disease of cattle worldwide. Although rare, it is imperative that the Canadian Limousin Association ensure that Protoporphyria status is recorded on the most prolific animals, therefore as of January 1, 2015, all donor cows in embryo production, must have a protoporphyria genotype on file at the Association. All females flushed prior to this date are grandfathered into the rules.

Protoporphyria testing can be done on-demand at any time with an approximate turnaround time of 2-4 weeks from the date the lab receives the sample, depending on whether it is done as a stand alone test (quicker) or in combination with genotyping. Testing must be requested through the CLA.

Horned/Polled & Coat Colour Testing

Horned/polled (homozygous polled) and coat colour (homozygous black) testing is available through the CLA (through our official laboratory Neogen Canada) and the results will automatically be reported into the DigitalBeef registry system and appear on the animal's registration certificate.

Genotypes by Pedigree

In general cases of simple recessive genes, when two homozygotes are mated together the resulting progeny will be homozygous for the trait. The Canadian Limousin Association uses this principle in regards to genetic abnormalities, color and polled traits.

a. Genetic Abnormalities: Progeny of animals that are designated as free of a genetic condition either by parentage or test are considered free of the condition in perpetuity due to the inherent low occurrence rate of those abnormalities in a population.b. Qualitative Traits: Progeny of animals that are tested homozygous for qualitative traits (i.e. black coat color, polled) are considered homozygous by pedigree. Subsequent progeny of homozygous by pedigree and/or homozygous by test animals will be designated homozygous by pedigree for three generations.a. Animals that are tested homozygous will designated with a “T” (ex. Homo Black (T))b. Animals designated homozygous by pedigree will be designated with a “P” and their generation (ex. Homo Black (P-1)). Homozygous by pedigree is dependent on the accuracy of an animal’s pedigree and the accuracy of the original tests in its pedigree.

What are gEPDs and what test should I choose?

DNA is the carrier of genetic information in all cellular forms of life, and is made up of small units called base pairs. Cattle have approximately 2.8 billion base pairs and humans have roughly 3 billion base pairs. There are four types of DNA base pairs, represented by the letters A, T, G, and C. It’s the order of the bases that make up the genetic code. A SNP, or Single Nucleotide Polymorphism, is a change in one of these base pairs. SNPs are normal in both cattle and humans, and contribute to the differences in appearance of each individual.

We know that each calf inherits half of its DNA from the mother and half from the sire. DNA testing can allow you to verify parentage, develop Molecular Breeding Values, and contribute to genomically enhanced Expected Progeny Differences (gEPDs). gEPDs are very similar to traditional EPDs and are used in the same manner. The primary difference is that gEPDs are much more accurate early in an animal’s life than traditional EPDs. This gives breeders the opportunity to make better breeding and selection decisions when an animal is young as opposed to when it already has many offspring, which may also be breeding at this time.

In order to generate gEPDs, two things are needed: 1. The traditional EPDs. Proper measurements of the traditional EPDs leads to increased accuracy, which contributes significantly to the accuracy of the prediction equations used to generate gEPDs for a breed. 2. Low or High Density SNP genotypes. The SNP genotypes are transformed into Molecular Breeding Values (MBVs). MBVs are then blended with the traditional EPDs to create gEPDs. In the Canadian Limousin Association’s Digital Beef system, gEPDs are highlighted yellow.

SNP testing in cattle can be broadly classified into three categories based on the number of SNPs that are assayed: basic parentage, low density, and high density SNP genotyping. 1. Basic SNP Parentage (200 SNPs)• The Basic SNP Parentage assay is a very good tool for parentage verification and has approximately the same accuracy as a microsatellite parentage test. • This assay does not collect enough information to be used for the generation of MBVs or gEPDs.2. Low Density SNP Genotyping (GGP uLD; 30,000 SNPs)• The GGP uLD assay is an excellent tool for conducting SNP parentage verification and is highly accurate. • The main advantage of the GGP uLD assay is that this information can be applied to the prediction equations for a specific breed to generate gEPDs at the lowest cost to the breeder.3. High Density SNP Genotyping (GGP 50K; 50,000 SNPs)• The Bovine 50K assay is also an excellent tool for conducting SNP parentage verification and is highly accurate.• Bovine 50K genotyping is used on highly prolific animals (i.e., AI sires, ET dams, and prolific herd sires) that contribute a great deal of genetic material to the entire herd through their many offspring. The Bovine 50K genotypes are used to develop the prediction equations for a specific breed’s gEPD calculation. Furthermore, by including animals from their herd, the prediction equations become that much more accurate for the breeder since their animals are contributing to the development of the calculations.• The Bovine 50K genotypes can also be applied to existing prediction equations to generate gEPDs on these animals.

Choose the test that reflects your needs as a producer. If you want to verify which bull is working overtime for you or sell progeny based on pedigree, then basic SNP parentage might be all you need. Going further, if you want to start incorporating genomics in your breeding plan, then a low-density test such as the GGP uLD is a good choice. If your business is breeding highly prolific animals, the Bovine 50K might do you the best service in terms of complete and accurate information. Which test you use depends on your on-farm breeding objective – this should help you meet your herd performance targets, and ultimately be based on profitability for the success and sustainability of your operation.

Transition from Microsatellite to SNP DNA Technology

We are now done with the conversion of our DNA bank to SNP. SNP is a state-of-the-art DNA testing method that is preferred around the world. We were able to obtain some funding to convert animals in our historical bank. Now that the funding has run out, any leftover animals with DNA on file can be converted at the expense of the CLA member.

We have come across some animals whose sample was not suitable for conversion (not enough hair follicles in most case). The progeny of animals that cannot be converted to SNP, but needing parentage verification, will be subject to double testing. Double testing means that we have to run a first test to verify the parents under the old method (microsatellite) as there is no other option. Then we run a second test by SNP so this new animal is on par with the rest of the population with results by SNP.

DNA transition explained by video

Leptin Testing

Leptin is a hormone that regulates feed intake, energy expenditure and whole body energy balance. A SNP in the Leptin gene has been associated with several economically important traits including milk production, weaning weight, backfat, marbling, quality grade, yield grade, dry matter intake, and days on feed. There are 3 Leptin variants:

CC (lean) animals have less fat and therefore, are less economically favourableCT animals are economically intermediateTT (fat) animals have more fat and therefore, are more economically favourable

The Canadian Limousin Association does not offer Leptin testing through the Association, however, the laboratory that independently offers these tests is listed below: